Methods for selecting eukaryotic cells expressing a heterologous protein

ABSTRACT

The invention pertains to a method for selecting at least one eukaryotic host cell expressing a product of interest, comprising
         (a) providing a plurality of eukaryotic host cells, wherein cellular viability of said host cells is dependent upon folate uptake, wherein said host cells comprise at least
           (i) a foreign polynucleotide encoding a product of interest and   (ii) a foreign polynucleotide encoding a DHFR enzyme;   
           (b) culturing said plurality of eukaryotic host cells in a selective culture medium comprising at least an inhibitor of DHFR and folate in a limiting concentration; and   (c) selecting at least one eukaryotic host cell expressing the product of interest.       

     Also provided is a method for expressing a product of interest which is based on host cells selected by said method and a cell culture medium.

FIELD OF THE INVENTION

The present invention relates to a novel method of selecting eukaryotichost cells, in particular mammalian host cells, expressing a product ofinterest. Furthermore, the present invention pertains to a method forefficiently producing a product of interest with a high yield.

BACKGROUND OF THE INVENTION

The ability to clone and express products of interest such asrecombinant peptides and proteins in large amounts has becomeincreasingly important. The ability to purify high levels of proteins isimportant in the human pharmaceutical and biotechnological field, forexample for producing protein pharmaceuticals as well as in the basicresearch setting, for example for crystallizing proteins to allow thedetermination of their three dimensional structure. Proteins that areotherwise difficult to obtain in quantity can be over-expressed in ahost cell and subsequently isolated and purified.

The choice of an expression system for the production of recombinantproteins depends on many factors, including cell growth characteristics,expression levels, intracellular and extracellular expression,post-translational modifications and biological activity of the proteinof interest, as well as regulatory issues and economic considerations inthe production of therapeutic proteins. Key advantages of mammaliancells over other expression systems such as bacteria or yeast are theability to carry out proper protein folding, complex N-linkedglycosylation and authentic O-linked glycosylation, as well as a broadspectrum of other post-translational modifications. Due to the describedadvantages, eukaryotic and in particular mammalian cells are currentlythe expression system of choice for producing complex therapeuticproteins such as monoclonal antibodies.

The most common approach to obtain high expressing host cells (alsocalled high producers) generates an appropriate expression vector forexpressing the product of interest as a first step. The expressionvector drives the expression of the polynucleotide encoding the productof interest in the host cell and provides at least one selectable markerfor generating the recombinant cell line. Key elements of mammalianexpression vectors usually include a constitutive or inducible promotercapable of robust transcriptional activity; optimized mRNA processingand translational signals that usually include a Kozak sequence, atranslation termination codon, mRNA cleavage and polyadenylationsignals, a transcription terminator and selectable markers for thepreparation of stable cell lines and for gene amplification; furthermorea prokaryotic origin of replication and selectable markers for vectorpropagation in bacteria can be provided by the expression vector.

In recent years the focus of development was concentrating on the designof improved vectors for gene expression in host cells. Despite of theplethora of available vectors, however, robust polypeptide/proteinproduction with a high yield in mammalian cells is still challenging.

One established procedure for obtaining high producing cell linesexpressing the product of interest with high yield is the stabletransfection of the host cells. However, the stable integration into thegenome is a rare event and only a small subset of stably transfectedcells are high producers. Their selection is accordingly challenging.

Selectable markers and selection systems are widely used in geneticengineering, recombinant DNA technology and the production ofrecombinant products in order to obtain host cells expressing theproduct of interest with high yield. Respective systems are also usefulto generate and identify stably transfected clones. The primary goal ofusing respective selectable markers and selection systems is tointroduce a selectable gene which upon exposure to selective growthconditions allows the identification of cells capable of high-levelproduction of the introduced selectable marker and accordingly, therecombinant product of interest. Increasing the yield of productexpression can be e.g. achieved by gene amplification using cells linese.g. deficient in an enzyme such as dihydrofolate reductase (DHFR) orglutamine synthetase (GS) in conjunction with expression vectorscontaining genes encoding these selectable marker enzymes and agentssuch as methotrexate (MTX), which inhibits DHFR, and methioninesulfoxamine (MSX) which inhibits GS.

One prominent selection system which is commonly used in the prior artis the dihydrofolate reductase/MTX selection system. Dihydrofolatereductase (DHFR) catalyzes the NADP-dependent reduction of dihydrofolicacid to tetrahydrofolic acid (THF). THF is then intraconverted to10-formyl-DHF and 5,10-methylene-DHF which are used in the de novobiosynthesis of purines and thymidylate, respectively. DHF is thebyproduct of the catalytic activity of thymidylate synthase (TS) whichcatalyzes the convertion of dUMP to dTMP in a 5,10-methylene-THFdependent reaction. Thus, DHFR is crucial for the recycling of THFcofactors that are essential for the biosynthesis of purine andpyrimidine nucleotides that are neccassary for the DNA replication.Hence, cells (for example CHO cells) that lack the DHFR gene (i.e. bytargeted genomic deletion) can be used as recipients for thetransfection of the DHFR gene in a medium that is free of nucleotides.After transfection, the cells can be subjected to gradual increase inthe concentrations of the antifolate MTX, a most potent DHFR inhibitor(Kd=1 pM), thereby forcing the cells to produce increased levels ofDHFR. After multiple rounds of selection, the selectable marker DHFRfrequently undergoes significant amplification. Also more sensitivemutant forms of the respective selectable markers can be used inconjunction with wildtype host cells. Alternatively, a mutant mouse DHFRwith a major resistance, i.e. less sensitivity, to MTX or other mutantforms of DHFR has also been extensively used as a dominant selectablemarker that markley enhanced the acquisition of high levelMTX-resistance in transfected cells. However, a major disadvantage ofthe DHFR/MTX selection system used in the prior art is that thistechnique utilizes a mutagenic cytotoxic agent, MTX, that canparticularly in higher concentrations alter the genotype of therecipients cells. This frequently results in MTX-resistant cellpopulations in which no expression of the target gene of interest ispresent due to loss of function mutations for example in the reducedfolate carrier (RFC)/or loss of RFC gene expression, both of whichabolish MTX uptake. However, increasing/high concentrations of MTX arenecessary, in order to achieve sufficiently stringent selectionconditions in order to isolate host cells producing the product ofinterest with a sufficient yield.

As becomes apparent, a high stringency selection system is crucial toenrich high producing cells from a transfected population. The higherthe stringency of the selection system the lower the number of lowproducers after the selection process and the higher the chance to findthe very rare ultra high producing clones in a transfected cellpopulation.

Therefore, it is the object of the present invention to provide astringent selection system for selecting host cells producing a productof interest with high yield, as well as methods for producing a productof interest with sufficient yield. In particular, it is the object ofthe present invention to provide a stringent selection system whichrequires less amounts of toxic agents, in particular MTX. Furthermore,it is the object of the present invention to provide a method forproducing a product of interest with a high yield.

SUMMARY OF THE INVENTION

The present invention pertains to a selection system for selecting hostcells expressing a product of interest with a high yield and to theproduction of respective products, in particular polypeptides such asantibodies.

According to one aspect, the present invention pertains to a method forselecting at least one eukaryotic host cell expressing a product ofinterest, said method comprising at least the following steps:

-   -   (a) providing a plurality of eukaryotic host cells, wherein the        cellular viability of said host cells is dependent upon folate        uptake, wherein said eukaryotic host cells comprise at least        -   (i) an introduced polynucleotide encoding a product of            interest and        -   (ii) an introduced polynucleotide encoding a DHFR enzyme;    -   (b) culturing said plurality of eukaryotic host cells in a        selective culture medium comprising at least an inhibitor of        DHFR and folate in a limiting concentration;    -   (c) selecting at least one eukaryotic host cell expressing the        product of interest.

The invention also relates to a process for producing a product ofinterest, comprising culturing a host cell selected according to thepresent invention under conditions that allow for the expression of theproduct of interest.

Also provided is a selective culture medium, comprising at least aninhibitor of DHFR and folate in a limiting concentration which can beused in the selection method according to the present invention. A“selective culture medium” is a cell culture medium useful for theselection of host cells.

Other objects, features, advantages and aspects of the presentapplication will become apparent to those skilled in the art from thefollowing description and appended claims. It should be understood,however, that the following description, appended claims, and specificexamples, while indicating preferred embodiments of the application, aregiven by way of illustration only. Various changes and modificationswithin the spirit and scope of the disclosed invention will becomereadily apparent to those skilled in the art from reading the following.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to a selection system using DHFR as aselectable marker, which requires a lower concentration of toxic agentssuch as the antifolate MTX but still provides stringent selectionconditions sufficient for identifying high producing host cells.

According to one aspect of the present invention, a method for selectingat least one eukaryotic host cell expressing a product of interest isprovided, said method comprising at least the following steps:

-   -   (a) providing a plurality of eukaryotic host cells, wherein        cellular viability of said host cells is dependent upon folate        uptake, wherein said eukaryotic host cells comprise at least        -   (i) an introduced polynucleotide encoding a product of            interest and        -   (ii) an introduced polynucleotide encoding a DHFR enzyme;    -   (b) culturing said plurality of eukaryotic host cells in a        selective culture medium comprising at least an inhibitor of        DHFR and folate in a limiting concentration;    -   (c) selecting at least one eukaryotic host cell expressing the        product of interest.

A “polynucleotide” is a polymer of nucleotides which are usually linkedfrom one deoxyribose or ribose to another and refers to DNA as well asRNA, depending on the context. The term “polynucleotide” does notcomprise any size restrictions and also encompasses polynucleotidescomprising modifications, in particular modified nucleotides.

A “product of interest” refers to the product to be expressed from saidhost cell. The product of interest may be e.g. a polypeptide or apolynucleotide, such as RNA. Preferably, the product of interest is apolypeptide, in particular an immunoglobulin molecule. Further examplesof products of interest are described in detail below.

An “introduced polynucleotide” refers to a polynucleotide sequence thathas been introduced into a host cell e.g. by the use of recombinanttechniques such as transfection. The host cell may or may not comprisean endogenous polynucleotide corresponding respectively being identicalto the introduced polynucleotide. Introduction may be achieved e.g. bytransfecting a suitable vector that may integrate into the genome of thehost cell (stable transfection). Suitable expression vectors allowingthe introduction of polynucleotides into the host cell are described indetail below. In case the heterologous nucleic acid is not inserted intothe genome, the heterologous nucleic acid can be lost at the later stagee.g. when the cells undergo mitosis (transient transfection). Suitablevectors might also be maintained in the host cell without integratinginto the genome, e.g. by episomal replication. However, also othertechniques are known in the prior art for introducing a polynucleotideinto a host cell which are described in further detail below.

An “inhibitor of DHFR” is a compound which inhibits the activity of thedihydrofolate reductase (DHFR). A respective inhibitor may for examplecompete with the DHFR substrate for binding to DHFR. Suitable DHFRinhibitors are for example antifolates such as methotrexate (MTX).Further examples include but are not limited to trimetrexate glucuronate(neutrexine), trimethoprim, pyrimethamine and pemetrexed.

The term “selecting” or “selection” as used herein, in particular refersto a process of using a selectable marker and selective culturingconditions to select and accordingly obtain host cells that haveincorporated the vector or vector combination according to the presentinvention. Thereby, successfully transfected host cells can be isolatedand/or enriched from the population of transfected host cells.

Host cells that have not successfully incorporated the vector or vectorcombination according to the present invention preferably die or areimpaired in growth under the selective culture conditions compared tohost cells that have successfully incorporated the vector or vectorcombination according to the present invention. During selection, hostcells which have successfully incorporated the vector or vectorcombination according to the present invention can be enriched as poolfrom the population of transfected host cells. Also individual hostcells can be isolated from the population of transfected host cellsduring selection (e.g. by clonal selection). Suitable embodiments ofselection procedures in order to obtain successfully transfected hostcells (e.g. by FACS sorting or limited dilution) are well known in theprior art and accordingly, need no detailed description.

A “limiting concentration of folate” refers to a concentration offolates in the selective culture medium which provides a selectivepressure on the host cell. Accordingly, folates are not comprised in theselective culture medium in affluence, thereby providing a selectionpressure on the host cells. The folate comprised in the selectiveculture medium in a limiting concentration is capable of being taken upinto and being processed by the host cell. Folates and in particularderivatives of folate which would not be processed by the host cellwould not contribute to the selection pressure and accordingly would notcontribute to the limiting concentration. Suitable concentration rangesare described below.

A “polypeptide” refers to a molecule comprising a polymer of amino acidslinked together by a peptide bond(s). Polypeptides include polypeptidesof any length, including proteins (for example, having more than 50amino acids) and peptides (for example, having 2-49 amino acids).Polypeptides include proteins and/or peptides of any activity orbioactivity. Suitable examples are outlined below.

It was surprisingly found that a selection system for providingrecombinant eukaryotic cells capable of producing a product of interestcan be based on the limited availability of folates in the selectiveculture medium in conjunction with the use of DHFR as selectable marker.The system is widely applicable, i.e. to eukaryotic host cells whichcellular viability depends on the uptake of folate. As is describedabove, the prior art must use rather high antifolate/MTX concentrationsin order to achieve a sufficient selection pressure for geneamplification and accordingly, to achieve an increase in the productionof the product of interest. This is a disadvantage as antifolates suchas MTX are toxic and may genetically alter the host cell. The approachof the present invention which is based on the combined use of a DHFRselection marker with a limiting concentration of folates in theselective culture medium has the advantage that the selection stringencyis considerable increased even at low DHFR inhibitor concentrations.Thus, when using the selection system of the present invention, highproducers are obtained even when using at low concentrations of the DHFRinhibitor (for example MTX) in the selective culture medium. Thus, lessDHFR inhibitor and accordingly less toxic agent concentrations areneeded when using the teaching of the present invention compared to theapproaches of the prior art for providing stringent selection conditionsthat allow the identification of high producing cell clones. Due to itsunique design, a very stringent selection system is provided allowingthe enrichment of high producing cells from the transfected host cellpopulation. This high stringency of the selection system according tothe present invention lowers the number of low producers in thepopulation after selection in the population and increases the chance tofind the very rare ultrahigh producing clones.

The selective culture medium may comprise one or more types of folate. Afolate according to the present invention can e.g. be an oxidized folate(i.e. folic acid) or a reduced folate or a derivative thereof. Ingeneral, a folate may be useful within the present invention as long assuch folate will be capable of being taken up into a eukaryotic cellpreferably by a functional membrane-bound folate receptor. The oxidizedfolate, i.e. folic acid, as well as reduced derivatives of folic acid,known as reduced folates or tetrahydrofolates (THF), are a group of B-9vitamins that are essential cofactors and/or coenzymes for thebiosynthesis of purines, thymidylate and certain amino acids ineukaryotic, in particular mammalian, cells. THF cofactors areparticularly crucial for DNA replication and hence cellularproliferation.

Specifically, THF cofactors function as donors of one-carbon units in aseries of interconnected metabolic pathways involving de novobiosynthesis of purines and thymidylate, amino acids as well as methylgroup metabolism, including CpG island methylation of DNA. Specifically,THF cofactors including 10-formyl-THF (10-CHO-THF) contribute one-carbonunits in two key de novo formyltransferase reactions involved in the denovo biosynthesis of purines. A preferred example of an oxidized folateis folic acid. Preferred examples of reduced folates are5-methyl-tetrahydrofolic acid, 5-formyl-tetrahydrofolic,10-formyl-tetrahydrofolic acid and 5,10-methylene-tetrahydrofolic acid.

The concentration of folate in the selective medium depends inparticular on the eukaryotic host cell used. A folate concentration of500 nM or less, 250 nM or less, 150 nM or less, 100 nM or less, 75 nM orless, 50 nM or less, 25 nM or less, 15 nM or less or even 10 nM or lesssuch as 7.5 nM or less is suitable. Suitable ranges include 0.1 nM-500nM, 0.1 nM-250 nM, 5 or 10 nM-250 nM, preferably 1 nM-150 nM, 5 or 10nM-150 nM, 1 nM-100 nM, 5 or 10 nM-100 nM and more preferred 1 nM-50 nM,2.5 nM-50 nM, 10 nM-50 nM or 12.5 nM-50 nM. These concentrations areparticularly suitable when using folic acid as folate.

Respective concentrations are limiting in the sense of the presentinvention and thus suitable to provide a selective pressure on the hostcells. The lower the concentration the stronger is the exerted selectionpressure as long as the cells are still viable. The describedconcentration ranges are particularly suitable for using CHO cells ashost cells.

The concentration of the DHFR inhibitor used in the selective culturemedium also depends on the eukaryotic host cell used. A DHFR inhibitorconcentration of 500 nM or less, 400 nM or less, 300 nM or less, 250 nMor less, 200 nM or less, 150 nM or less is advantageous in case theconcentration of the DHFR inhibitor in the selective medium is supposedto be reduced. However, preferably, the selective medium comprises atleast 10 nM of the DHFR inhibitor. Preferred antifolate concentrations,preferably MTX, are 1 nM-500 nM, preferably 10 nM-200 nM, 10 nM-150 nMand more preferred 10 nM-100 nM. Respective concentrations in theselective culture medium are particularly suitable for CHO cells.

The preferred concentrations and concentration ranges of folate andantifolate described above can be combined with each other. According toone embodiment, a selective culture medium is used which comprises aconcentration of DHFR inhibitor, preferably MTX, of 200 nM or less,preferably 150 nM or less, preferably 100 nM or less and a folateconcentration, preferably folic acid, of less than 100 nM, preferablyless than 75 nM. In one embodiment, a folate concentration of 12.5 nM-50nM is used in combination with an antifolate concentration of 10 nM-100nM. Preferably, folic acid and MTX are used as folate and antifolate.

The feasible concentrations of folic acid and MTX may be dependent fromone another; a preferred combination is a concentration of folic acid at2.5 nM-75 nM, 2.5 nM-50 nM or 12.5 nM-50 nM combined with aconcentration of MTX at 10 nM-500 nM, preferably 10 nM-100 nM. Theseconcentrations are particularly preferred when using a DHFR+ (plus)cell.

The concentrations described above are particularly suitable for fastgrowing suspension cells, which is a preferred phenotype for commercialproduction cell lines. However, different cell lines may have differentfolic acid consumption properties. Furthermore, the limiting/selectiveconcentrations may vary depending on the used folate, respectivelyantifolate. Therefore, the limiting concentrations of folate, inparticular folic acid and antifolate, in particular MTX as well as thesuitable folic acid to MTX ratios may differ for different cell lines.Suitable concentrations, however, can easily be determinedexperimentally by the skilled person.

According to one embodiment, the host cells are pre-cultured in a folatefree culture medium or in a culture medium comprising a limitingconcentration of folate prior to transfection and/or selection. Suitablelimiting concentrations of folate are described above. Preferably, saidculture medium for pre-culturing the host cells comprises folate, inparticular folic acid in a concentration of 50 nM or less.

The expression of the incorporated selectable marker DHFR provides aselective advantage under selective culture conditions to the hostcells. E.g. host cells (e.g. CHO cells) that lack the DHFR gene (e.g. bytargeted genomic deletion, also called DHFR⁻ host cells) can be used asrecipients for the transfection of the DHFR gene as selectable markergene in a medium that is free of nucleotides. However, it is alsopossible to use host cells that express DHFR endogenously (DHFR⁺ (plus)host cells) when performing a DHFR selection, if appropriate selectiveculture conditions are used. After transfection with the polynucleotidesaccording to the present invention, the cells can be subjected to agradual increase in the concentrations of inhibitors of DHFR. Oneexample of DHFR inhibitors are antifolates such as MTX, which is apotent DHFR inhibitor (Kd=1 pM). The presence of the antifolate such asMTX in the medium forces the cells to produce increased levels of DHFRin order to survive. Upon multiple rounds of selection, the selectablemarker DHFR frequently undergoes significant gene amplification in orderto achieve that.

Several suitable DHFR genes are known in the prior art that can be usedin conjunction with the present invention. The DHFR may be a wildtypeDHFR or a functional variant or derivative thereof. The term a “variant”or “derivative” include DHFR enzymes having one or more amino acidsequence exchanges (e.g. deletions, substitutions or additions) withrespect to the amino acid sequence of the respective DHFR enzyme, fusionproteins comprising a DHFR enzyme or functional fragment thereof andDHFR enzymes which have been modified to provide an additional structureand/or function, as well as functional fragments of the foregoing, whichstill have at least one function of a DHFR enzyme. DHFR enzymes/variantscan be used as selection marker, which are more or less sensitive to MTXthan the wildtype DHFR enzyme. According to one embodiment, the DHFRenzyme used is more sensitive for antifolates such as MTX than thecorresponding wildtype DHFR enzyme and/or the DHFR enzyme endogenouslyexpressed by the host cell if expressed. The DHFR enzyme can be derivedfrom any species as long as it will be functional within the presentinvention, i.e. compatible with the host cell utilised. E.g., a mutantmouse DHFR with a major resistance to MTX has been extensively used as adominant selectable marker that markedly enhances the acquisition ofhigh level MTX-resistance in transfectant cells. Preferably, a DHFRenzyme is used which is less susceptible and thus less sensitive to aDHFR inhibitor such as MTX than the DHFR enzyme endogenously expressedin a DHFR⁺ (plus) host cell.

According to one embodiment, an intron or a fragment thereof is placedat the 3′ end of the open reading frame of the DHFR gene. This hasadvantageous effects on the expression/amplification rate of theconstruct. The intron used in the DHFR expression cassette is leading toa smaller, non functional variant of the DHFR gene (Grillari et al.,2001, J. Biotechnol. 87, 59-65). Thereby the expression level of theDHFR gene is lowered and can thus further increase the stringency of theselection system. Accordingly, the host cell may comprise an introducedpolynucleotide encoding a DHFR enzyme, said polynucleotide comprising anintron which is located 3′ of the DHFR coding sequence. Alternativemethods making use of an intron to reduce the expression level of theDHFR gene are described in EP 0 724 639 and could also be used.

In contrast to most prokaryotes, plants and fungi which synthesize theirown folates, mammals and other eukaryotic species are devoid of THFcofactor biosynthesis and must therefore obtain them from exogenoussources, usually the culture medium. Three independent transport systemsare currently known to mediate the uptake of folates and antifolates inmammalian cells, namely the reduced folate carrier (RFC); theproton-coupled folate transporter (PCFT, also known as SLC46A) andfolate receptors (FRs).

The eukaryotic host cell is, preferably, selected from the groupconsisting of a mammalian cell, an insect cell, a plant cell and a fungicell. Fungi cells and plant cells can be prototrophic for folates (i.e.such cells can autonomously synthesize their own folates necessary fortheir cellular viability, i.e. cellular growth and proliferation). Thepresent invention encompasses in particular such fungi and plant cellswhich are or may become auxotrophic for folates. This may be for exampledue to genetic manipulation, i.e. cells are now unable to synthesizesufficient amounts of folates necessary for their cellular viability.For example, the capacity of such fungi or plant cells to endogenouslybiosynthesize folates, e.g. via an appropriate metabolic pathway, can beinactivated, e.g. by gene disruption or gene silencing of appropriatetarget genes, or inhibition of key enzymes, etc. Preferably, the hostcell is a mammalian cell. Said mammalian cell can be selected from thegroup consisting of a rodent cell, a human cell and a monkey cell.Particularly preferred is a rodent cell, which preferably is selectedfrom the group consisting of a CHO cell, a BHK cell, a NSO cell, a mouse3T3 fibroblast cell, and a SP2/0 cell. A most particularly preferredrodent cell is a CHO cell. Also preferred is a human cell, which,preferably, is selected from the group consisting of a HEK293 cell, aMCF-7 cell, a PerC6 cell, and a HeLa cell. Further preferred is a monkeycell, which, preferably, is selected from the group consisting of aCOS-1, a COS-7 cell and a Vero cell. The host cell is preferably a DHFR⁺(plus) cell, in particular a DHFR⁺ (plus) CHO cell.

According to one embodiment, the polynucleotide encoding a product ofinterest and the polynucleotide encoding a DHFR enzyme were introducedby at least one expression vector. Suitable techniques for introducing arespective vector are described below and include e.g. transfection.

An “expression vector” according to the present invention is apolynucleotide capable of carrying at least one foreign nucleic acidfragment. A vector functions like a molecular carrier, deliveringfragments of nucleic acids respectively polynucleotides into a hostcell. It comprises at least one expression cassette comprisingregulatory sequences for properly expressing a polynucleotideincorporated therein. Polynucleotides (e.g. encoding the product ofinterest or selectable markers) may be inserted into the expressioncassette(s) of the expression vector in order to be expressed therefrom.The expression vector according to the present invention may be presentin circular or linearized form. The term “expression vector” alsocomprises artificial chromosomes or similar respective polynucleotidesallowing the transfer of foreign nucleic acid fragments.

The polynucleotide encoding the product of interest and thepolynucleotide encoding the DHFR enzyme can be located on the same ordifferent expression vectors. Using an expression vector carrying bothpolynucleotides has the advantage that only one expression vector needsto be introduced into the host cell. Furthermore, in particular whenestablishing a stable expression line it is more likely that thepolynucleotides are integrated together into the genome and,accordingly, expressed with a similar yield. However, it is alsopossible and within the scope of the present invention to use acombination of at least two expression vectors for transfection, whereinthe respective polynucleotides are located on different expressionvectors. Said combination of expression vectors is then transfected intothe host cell.

The eukaryotic host cell may comprise at least one additional introducedpolynucleotide encoding a further product of interest. This embodimentis particularly suitable for expressing immunoglobulin molecules.According to a preferred embodiment, the host cell comprises at leasttwo introduced polynucleotides each encoding a product of interest,wherein at least one polynucleotide encodes the heavy chain of animmunoglobulin molecule or a functional fragment thereof and the otherpolynucleotide encodes the light chain of an immunoglobulin molecule ora functional fragment thereof. The respective polynucleotides can beintroduced by using an appropriate expression vector. Saidpolynucleotides encoding the heavy and light chain of an immunoglobulinmolecule (or a functional fragment thereof) may be located on the sameor on different expression vectors in case a combination of at least twoexpression vectors is used.

The host cell and accordingly the expression vector for introducingpolynucleotides into said host cell may additionally comprise one ormore further polynucleotide(s) encoding one or more additionalselectable marker(s). Accordingly, in one embodiment of the presentinvention co-selection utilizing the system of the present inventiontogether with one or more different selection system(s) (e.g. antibioticresistant selection systems such as neo/G418) can be applied to furtherimprove the performance. Besides further eukaryotic selectable markers,allowing the selection of eukaryotic host cells, also prokaryoticselectable markers can be used, which allow the selection in eukaryotichost cells. Examples of respective prokaryotic selectable markers aremarkers which provide a resistance to antibiotics such as e.g.ampicillin, kanamycin, tetracycline and/or chloramphenicol.

Vectors used for introducing the polynucleotides into the host cellsusually contain transcriptional control elements suitable to drivetranscription such as e.g. promoters, enhancers, polyadenylationsignals, transcription pausing or termination signals as element of anexpression cassette. If the desired product is a protein, suitabletranslational control elements are preferably included in the vector andoperably linked to the polynucleotides to be expressed, such as e.g. 5′untranslated regions leading to 5′ cap structures suitable forrecruiting ribosomes and stop codons to terminate the translationprocess. In particular, the polynucleotide serving as the selectablemarker genes as well as the polynucleotide encoding the product ofinterest can be transcribed under the control of transcription elementspresent in appropriate promoters. The resultant transcripts of theselectable marker genes and that of the product of interest harbourfunctional translation elements that facilitate substantial levels ofprotein expression (i.e. translation) and proper translationtermination. A functional expression unit, capable of properly drivingthe expression of an incorporated polynucleotide is also referred to asan “expression cassette” herein.

The expression vector or combination of expression vectors according tothe present invention used for introducing the polynucleotides into theeukaryotic host cells may comprise at least one promoter and/orpromoter/enhancer element as element of an expression cassette. Althoughthe physical boundaries between these two control elements are notalways clear, the term “promoter” usually refers to a site on thenucleic acid molecule to which an RNA polymerase and/or any associatedfactors binds and at which transcription is initiated. Enhancerspotentiate promoter activity, temporally as well as spatially. Manypromoters are transcriptionally active in a wide range of cell types.Promoters can be divided in two classes, those that functionconstitutively and those that are regulated by induction orderepression. Both classes are suitable for the teachings of the presentinvention. Promoters used for high-level production of polypeptides inmammalian cells should be strong and preferably active in a wide rangeof cell types.

Strong constitutive promoters which drive expression in many cell typesinclude but are not limited to the adenovirus major late promoter, thehuman cytomegalovirus immediate early promoter, the SV40 and RousSarcoma virus promoter, and the murine 3-phosphoglycerate kinasepromoter, EF1a. Good results are achieved with the expression vector ofthe present invention when the promoter and/or enhancer is eitherobtained from CMV and/or SV40. The transcription promoters can beselected from the group consisting of an SV40 promoter, a CMV promoter,an EF1alpha promoter, a RSV promoter, a BROAD3 promoter, a murine rosa26 promoter, a pCEFL promoter and a β-actin promoter.

Preferably, the polynucleotide encoding the product of interest and thepolynucleotide encoding the DHFR enzyme are under the control ofdistinct transcription promoters. In general, a promoter capable ofpromoting expression, in particular transcription, of the essentialpolynucleotides in a eukaryotic host cell will be suitable. The distincttranscription promoters driving the expression from the polynucleotidescan be the same or different.

According to one embodiment, a stronger promoter and/or enhancer is usedfor driving the expression of the polynucleotide encoding the product ofinterest than for driving the expression of the polynucleotide encodingthe DHFR enzyme and/or the additional selectable markers if present.This arrangement has the effect that more transcript is generated forthe product of interest than for the selectable markers. It isadvantageous that the production of the product of interest is dominantover the production of the selectable markers, since the individual cellcapacity for producing heterologous products is not unlimited and shouldthus be focused to the product of interest. Furthermore, the selectionprocess only occurs at the initial stages of establishing an expressioncell line, which then constantly produces the product of interest. Thus,it is advantageous to focus the resources of the cells to theexpression/production of the product of interest. Furthermore, using aless strong promoter for expressing the selectable marker(s), inparticular DHFR, further increases the selection pressure and thusallows the use of lower concentrations of DHFR inhibitors in theselective culture medium.

According to one embodiment, the promoter driving the expression of thepolynucleotide encoding the product of interest is a CMV promoter andthe promoter driving the expression of the polynucleotide encoding theDHFR enzyme is a SV40 promoter. The CMV promoter is known to be one ofthe strongest promoters available for mammalian expression and leads toa very good expression rate. It is considered to give significantly moretranscript than the SV40 promoter.

According to a further embodiment, the polynucleotide encoding theproduct of interest and the polynucleotide encoding the DHFR enzyme areunder the control of the same transcription promoter and are thusexpressed from one expression cassette. Suitable promoters are describedabove. In this embodiment, one long transcript is obtained from therespective expression cassette that is under the control of saidtranscription promoter. According to one embodiment, at least one IRESelement is functionally located between the polynucleotide encoding theproduct of interest and/or the polynucleotide encoding the DHFR enzyme.Thereby, it is ensured that separate translation products are obtainedfrom said transcript.

The expression cassette may comprise an appropriate transcriptiontermination site. This, as continued transcription from an upstreampromoter through a second transcription unit may inhibit the function ofthe downstream promoter, a phenomenon known as promoter occlusion ortranscriptional interference. This event has been described in bothprokaryotes and eukaryotes. The proper placement of transcriptionaltermination signals between two transcription units can prevent promoterocclusion. Transcription termination sites are well characterized andtheir incorporation in expression vectors has been shown to havemultiple beneficial effects on gene expression.

The host cell used is a eukaryotic, in particular a mammalian host cell.Most eukaryotic nascent mRNAs possess a poly A tail at their 3′ endwhich is added during a complex process that involves cleavage of theprimary transcript and a coupled polyadenylation reaction. The polyAtail is advantageous for mRNA stability and transferability. Hence, theexpression cassettes for expressing the polynucleotides encoding theproduct of interest and the DHFR enzyme usually comprise apolyadenylation site suitable for transcription termination andpolyadenylation. There are several efficient polyA signals that can beused in mammalian expression vectors, including those derived frombovine growth hormone (bgh), mouse beta-globin, the SV40 earlytranscription unit and the Herpes simplex virus thymidine kinase gene.However, also synthetic polyadenylation sites are known (see e.g. thepCl-neo expression vector of Promega which is based on Levitt el al,1989, Genes Dev. 3, (7): 1019-1025). The polyadenylation site can beselected from the group consisting of SV40polyA site, such as the SV40late and early poly-A site (see e.g. plasmid pSV2-DHFR as described inSubramani et al, 1981, Mol. Cell. Biol. 854-864), a synthetic polyA site(see e.g. the pCl-neo expression vector of Promega which is based onLevitt el al, 1989, Genes Dev. 3, (7): 1019-1025) and a bgh polyA site(bovine growth hormone).

Furthermore, an expression cassette comprising the polynucleotideencoding the product of interest and the polynucleotide encoding theDHFR enzyme may comprise at least one intron. This embodiment isparticularly suitable when a mammalian host cell is used for expression.Most genes from higher eukaryotes contain introns which are removedduring RNA processing. Respective constructs are expressed moreefficiently in transgenic systems than identical constructs lackingintrons. Usually, introns are placed at the 5′ end of the open readingframe but may also be placed at the 3′ end. Accordingly, an intron maybe comprised in the expression cassette(s) to increase the expressionrate. Said intron may be located between the promoter and orpromoter/enhancer element(s) and the 5′ end of the open reading frame ofthe polynucleotide to be expressed. Several suitable introns are knownin the state of the art that can be used in conjunction with the presentinvention.

According to one embodiment, the intron used in the expression cassettesfor expressing the product of interest, is a synthetic intron such asthe SIS or the RK intron. The RK intron is a strong synthetic intronwhich is preferably placed before the ATG start codon of the gene ofinterest. The RK intron consists of the intron donor splice site of theCMV promoter and the acceptor splice site of the mouse IgG Heavy chainvariable region (see e.g. Eaton et al., 1986, Biochemistry 25,8343-8347, Neuberger et al., 1983, EMBO J. 2(8), 1373-1378; it can beobtained from the pRK-5 vector (BD PharMingen)).

An expression vector comprising the polynucleotides and/or expressioncassettes as described above can be transfected into the host cell inits circular form. Supercoiled vector molecules usually will beconverted into linear molecules within the nucleus due to the activityof endo- and exonucleases. However, linearization of the expressionvector before transfection often improves the efficiency of a stabletransfection. This also as the point of linearization may be controlledif the expression vector is linearized prior to transfection. Hence,according to one embodiment of the present invention the expressionvector or combination of at least two expression vectors comprises atleast one predefined restriction site, which can be used forlinearization of the vector(s) prior to transfection. According to oneembodiment, the linearization site is arranged such, that uponlinearization, the polynucleotide encoding the DHFR enzyme is located 5′of the polynucleotide encoding the product of interest. This arrangementis advantageous for gene amplification. In case a prokaryotic selectablemarker is additionally used, the polynucleotide encoding saidprokaryotic marker is located 3′ of the polynucleotide encoding theproduct of interest. This has the effect that the prokaryotic selectionmarker gene is 3′ and thus “outside” of the “mammalian” parts of thelinearized vector nucleic acid. This arrangement is favourable sinceprokaryotic genes are presumably not advantageous for mammalianexpression as prokaryotic sequences may lead to increased methylation orother silencing effects in the mammalian cells.

The polynucleotide encoding a product of interest and the polynucleotideencoding the DHFR enzyme are preferably stably introduced into said hostcell. The stable introduction respectively transfection is advantageousfor establishing of expression cell lines and in particular for thelarge scale and accordingly industrial production of the product ofinterest.

There are several appropriate methods known in the prior art forintroducing polynucleotides and expression vectors into eukaryotic hostcells, including mammalian host cells. Respective methods include butare not limited to calcium phosphate transfection, electroporation,lipofection, biolistic- and polymer-mediated genes transfer. Besidestraditional random integration based methods also recombination mediatedapproaches can be used to transfer the polynucleotide encoding theproduct of interest and the polynucleotides encoding a DHFR enzyme intothe host cell genome. Such recombination methods may include use of sitespecific recombinases like Cre, Flp or ΦC31 (see e.g. Oumard et al,Cytotechnology (2006) 50: 93-108) which can mediate directed insertionof transgenes. Alternatively, the mechanism of homologous recombinationmight be used to insert said polynucleotides (reviewed in Sorrell et al,Biotechnology Advances 23 (2005) 431-469). Recombination based geneinsertion allows to minimize the number of elements to be included inthe heterologous nucleic acid that is transferred/introduced to the hostcell. For example, an insertion locus might be used that alreadyprovides promoter and poly-A site (exogenous or endogenous) such thatonly the remaining elements (e.g. the polynucleotide encoding theproduct of interest and the polynucleotide encoding the DHFR enzymeneeds to be transferred/transfected to the host cell. Embodiments of asuitable expression vector or combination of expression vectorsaccording to the present invention as well as suitable host cells aredescribed in detail above; we refer to the above disclosure.

In case a further selectable marker is used in addition to the DHFRenzyme, the selective conditions for said selectable marker can beapplied prior to applying the selective conditions for the DHFR enzyme.E.g. in case the neomycin phosphotransferase gene (neo) is used asadditional selectable marker, the cells can be grown first in a mediume.g. containing G418 in order to select cells that have incorporated theexpression vector(s) according to the present invention.

The strategy of the present invention to use a limiting concentration offolate in the selective culture medium in addition to a DHFR selectablemarker has the advantage that a very high stringency is obtained even iflower DHFR inhibitor concentrations are used. The productivity of thecell population surviving these novel selection conditions is remarkablyincreased. The examples have shown that the host cells obtained afterthe selection method produce the product of interest with a high yield.Also the average productivity of the individual host cells is increased.Thus, chances are improved to find high producer clones with lowerscreening efforts. Thus, the selection system according to the presentinvention is superior to selection systems used in the prior art. Inparticular host cells are obtained, which have a higher productivitycompared to the use of the respective selectable markers alone. Thus,due to the higher stringency of the selection conditions, the selectionprocedure is optimized.

Cells obtained as a result of the stringent screening/selectionprocedure of the present invention will generally be isolated and may beenriched from non-selected cells of the original cell population. Theycan be isolated and cultured as individual cells. They can also be usedin one or more additional rounds of selection, optionally for additionalqualitative or quantitative analysis, or can be used e.g. in developmentof a cell line for protein production. According to one embodiment, anenriched population of producing host cells selected as described aboveis directly used as population for the production of the polypeptide ofinterest with a good yield. Preferably, a host cell is selected whichstably expresses the product of interest. The advantages of a stabletransfection/expression are described in detail above. We refer to theabove disclosure.

Also provided is a method for producing a product of interest,comprising at least the following steps:

-   -   (a) performing the selection method according to the present        invention for selecting at least one eukaryotic host cell        expressing the product of interest; and    -   (b) culturing at least one selected eukaryotic host cell under        conditions that allow for the expression of the product of        interest.

As the selection method according to the present inventions allows theidentification of high producing cell clones, said selection system isan important and integral part of the production process. The expressedproduct of interest may be obtained by disrupting the host cells. Thepolypeptides may also be expressed, e.g. secreted into the culturemedium and can be obtained therefrom. Also combinations of therespective methods are possible. According to one embodiment, said hostcells are cultured under serum-free conditions.

Thereby, products, in particular polypeptides, can be produced andobtained/isolated efficiently with high yield. The obtained product mayalso be subject to further processing steps such as e.g. purificationand/or modification steps. Accordingly, the method for producing theproduct of interest may comprise at least one of the following steps:

-   -   isolating the product of interest from said cell culture medium        and/or from said host cell; and/or    -   processing the isolated product of interest.

The product of interest, for example a polypeptide, produced inaccordance with the invention may be recovered and optionally furtherprocessed, e.g. further purified, isolated and/or modified by methodsknown in the art. For example, the product may be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, ultra-filtration, extraction orprecipitation. Purification may be performed by a variety of proceduresknown in the art including, but not limited to, chromatography (e.g. ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g. ammonium sulfate precipitation) orextraction.

The product of interest can be any biological product capable of beingproduced by transcription, translation or any other event of expressionof the genetic information encoded by said polynucleotide. In thisrespect, the product will be an expression product. The product ofinterest may be selected from the group consisting of polypeptides,nucleic acids, in particular RNA or DNA. The product can be apharmaceutically or therapeutically active compound, or a research toolto be utilized in assays and the like. In a particularly preferredembodiment, the product is a polypeptide, preferably a pharmaceuticallyor therapeutically active polypeptide, or a research tool to be utilizedin diagnostic or other assays and the like. A polypeptide is accordinglynot limited to any particular protein or group of proteins, but may onthe contrary be any protein, of any size, function or origin, which onedesires to select and/or express by the methods described herein.Accordingly, several different polypeptides of interest may beexpressed/produced. As is outlined above, the term polypeptides includeproteins and/or peptides of any activity or bioactivity, including e.g.bioactive polypeptides such as enzymatic proteins or peptides (e.g.proteases, kinases, phosphatases), receptor proteins or peptides,transporter proteins or peptides, bactericidal and/or endotoxin-bindingproteins, structural proteins or peptides, immune polypeptides, toxins,antibiotics, hormones, growth factors, vaccines or the like. Saidpolypeptide may be selected from the group consisting of peptidehormones, interleukins, tissue plasminogen activators, cytokines,immunoglobulins, in particular antibodies or functional antibodyfragments or variants thereof. In a most preferred embodiment thepolypeptide is an immunoglobulin molecule or antibody, or a functionalvariant thereof, for example a chimeric, or a partly or totallyhumanized antibody. Such an antibody can be a diagnostic antibody, or apharmaceutically or therapeutically active antibody.

Also provided is a product obtained by a method according to the presentinvention as defined above and in the claims. Said product is preferablya polypeptide, in particular an immunoglobulin molecule or a functionalfragment thereof.

According to one embodiment, the present invention also provides aselective culture medium comprising folate in a limiting concentrationand at least one inhibitor of DHFR. Preferably, said selective culturemedium has one or more of the following characteristics:

-   -   (a) it comprises folate, preferably folic acid, in a        concentration selected from:        -   (aa) 500 nM or less;        -   (bb) 250 nM or less;        -   (cc) 150 nM or less;        -   (dd) 100 nM or less;        -   (ee) 75 nM or less;        -   (ff) 50 nM or less;        -   (gg) 25 nM or less, and/or        -   (hh) 15 nM or less;    -   and/or    -   (b) it comprises folate, preferably folic acid, in a        concentration range selected from        -   (aa) 0.1 nM-500 nM;        -   (bb) 0.1 nM-250 nM, preferably 2.5 nM-250 nM or 5 or 10            nM-250 nM;        -   (cc) 0.1 nM-150 nM, preferably 2.5 nM-150 nM or 5 or 10            nM-150 nM;        -   (dd) 1 nM-100 nM; preferably 2.5 nM-100 nM or 5 or 10 nM-100            nM;        -   (ee) 1 nM-75 nM; preferably 2.5 nM-75 nM or 5 or 10 nM-75            nM;        -   (ff) 1 nM-50 nM;        -   (gg) 2.5 nM-50 nM; and/or        -   (hh) 12.5 nM-50 nM    -   and/or    -   (c) it comprises the DHFR inhibitor, which is preferably an        antifolate, in a concentration selected from        -   (aa) 500 nM or less;        -   (bb) 400 nM or less;        -   (cc) 300 nM or less;        -   (dd) 250 nM or less;        -   (ee) 200 nM or less;        -   (ff) 150 nM or less; and/or        -   (gg) 100 nM or less;    -   and/or    -   (d) it comprises the DHFR inhibitor, which is preferably an        antifolate and more preferred MTX, in a concentration selected        from        -   (aa) 1 nM-500 nM;        -   (bb) 10 nM-200 nM;        -   (cc) 10 nM-150 nM; and/or        -   (dd) 10 nM-100 nM.

The indicated concentrations and concentration ranges for the folate andthe DHFR inhibitor can be combined with each other. The advantages andfurther preferred embodiments of concentration ranges and suitableembodiments for folates and antifolates in the selective culture mediumwere outlined in detail above in conjunction with the selection methodaccording to the present invention; it is referred to the abovedisclosure. Said selective culture medium can be used in conjunctionwith the selection system of the present invention.

The full contents of the texts and documents as mentioned herein areincorporated herein by reference and thus form part of the presentdisclosure.

The following examples serve to illustrate the present invention withoutin any way limiting the scope thereof. In particular, the examplesrelate to preferred embodiments of the present invention.

EXAMPLES

In general, suitable materials, such as reagents, are familiar to theskilled person, commercially available and can be used in accordancewith the manufacturer's instructions. The experiments were performed asdescribed.

A transfection experiment in CHO cells is done using an expressionvector containing expression cassettes for expressing a monoclonalantibody as product of interest. As selectable markers, a G418resistence gene (NEO) and a DHFR gene are present on the expressionvector in separate expression cassettes. This experiment demonstratesthat the selection with reduced MTX amounts under low folic acidconditions yields high producing cell populations. As reference,standard selection conditions for DHFR are used, which use higher MTXconcentrations and folic acid in non-limiting amounts.

Example I DHFR and Limiting Concentrations of Folic Acid

1.1. The Expression Vector

The expression vector is a mammalian expression vector comprising thefollowing decisive elements, which are arranged in the same orientationon the expression vector:

CMV promoter/enhancer Intron Polynucleotide encoding the antibody lightchain Multiple cloning site SV40 poly A site CMV promoter/enhancerIntron Polynucleotide encoding the antibody heavy chain Multiple cloningsite SV40 poly A site SV40 enhancer/promoter Neomycin phosphotransferase(neo) Poly A site (synthetic) Ampicillin resistance gene SV40 promoterDHFR mutant gene being less sensible to MTX than the DHFR wildtypeIntron Poly A site

1.2. Transfection and Selection of CHO-Cells

Cell cultivation, transfection and screening is carried out in shakeflasks using a suspension of growing CHO cells in a culture mediumappropriate for CHO cells without FCS. Cells were transfected with theexpression vector by electroporation. In order to reduce intracellularfolic acid reservoirs in the host cells and to prevent co-transfer offolic acid from the pre-culture medium to the selection medium, cellsare passaged to folic acid free medium or medium with reduced folic acidcontent (e.g. 50 nM) prior to the transfection and selection. Dependingon the cell viability, a first selection step is started 24-48 h aftertransfection by adding G418 and MTX containing selective culture mediumto the cells. In a first selection step, three different MTX (2.5, 5 and10 nM) and folic acid concentrations (12.5, 25 and 50 nM) are tested. Asreference, a culture medium is used comprising non-limiting amounts offolic acid, here 11.3 μM—which corresponds to a standard concentrationin the culture medium).

As soon as cells recover to a viability of above 80%, a second selectionstep is applied by passaging the cells to G418 free medium containingthe same amount of folic acid as in the first selection step but 10times as much MTX (i.e. 25, 50 and 100 nM). In case of the referenceculture conditions, 500 nM MTX is added to the cells.

1.3. Determination of Pool Productivity

Productivity of the selected cell populations is analyzed after thefirst and final selection steps via overgrown shake flask batch culturesin a medium containing non-limiting amounts of folic acid (11.3 μM)without G418, but containing MTX in the same concentration as in therespective selection medium.

Batch cultures are seeded in a shake flask having 250 mL capacity with50 mL working volume and are cultivated in a shaker cabinet (nothumidified) at 150 rpm and 10% CO2. Viability of cells have to be >90%when starting the assay. The seeding cell density is usually about 2×10⁵c/mL. Titer determination takes place at day 13. Antibody titers in thecell culture supernatant are determined by protein-A HPLC 13 days afterstarting the culture.

1.4. Results

To evaluate the stringency of dhfr/MTX selection under limiting folicacid concentrations, transfection of a DHFR vector expression forexpressing a monoclonal antibody is done in this example I. The vectoralso contains a G418 resistance gene (see above). First, transfectedcell populations are selected by adding G418 and differentconcentrations of MTX at different concentrations of folic acid. Thisinitial first selection step should help to kill untransfected cells andin parallel force the cells to consume intracellular folic acidreservoirs before higher stringency is applied in the second selectionstep. Under these conditions all transfected cell populations usuallyrecovered and productivity is assessed as described above.

Table 1 summarizes the productivity results obtained:

TABLE 1 Productivity of cell populations after 1^(st) selection step.Productivity Selection Medium mAb (mg/L) I. Test series without (w/o)MTX and low folic acid (FA) concentrations (12.5 nM, 25 nM and 50 nM)+0.8 g/L G418 16 +12.5 nM FA +w/o MTX +0.8 g/L G418 12 +25 nM FA +w/oMTX +0.8 g/L G418 17 +50 nM FA +w/o MTX II. Test series with a low MTXconcentration (2.5 nM) and low folic acid (FA) concentrations (12.5 nM,25 nM and 50 nM) +0.8 g/L G418 12 +12.5 nM FA +2.5 nM MTX +0.8 g/L G41817 +25 nM FA +2.5 nM MTX +0.8 g/L G418 11 +50 nM FA +2.5 nM MTX III.Test series with a low MTX concentration (5 nM) and low folic acid (FA)concentrations (12.5 nM, 25 nM and 50 nM) +0.8 g/L G418 12 +12.5 nM FA+5 nM MTX +0.8 g/L G418 33 +25 nM FA +5 nM MTX +0.8 g/L G418 16 +50 nMFA +5 nM MTX IV. Test series with a low MTX concentration (10 nM) andlow folic acid (FA) concentrations (12.5 nM, 25 nM and 50 nM) +0.8 g/LG418 205 +12.5 nM FA +10 nM MTX +0.8 g/L G418 30 +25 nM FA +10 nM MTX+0.8 g/L G418 23 +50 nM FA +10 nM MTX V. Test series with non-limitingconcentrations of folic acid (FA) (11.3 μM) and different MTXconcentrations (0; 2; 2.5; 5 and 10 nM) +0.8 g/L G418 16 +11.3 μM FA+w/o MTX +0.8 g/L G418 11 +11.3 μM FA +2.5 nM MTX +0.8 g/L G418 16 +11.3μM FA +5 nM MTX +0.8 g/L G418 11 +11.3 μM FA +10 nM MTX

Transfected cells selected in G418 and MTX containing medium withdifferent folic acid concentrations are analyzed in shake flask batchcultures. At day 13 of the culture, samples of the culture medium aretaken and are analyzed for antibody content by Protein-A HPLC.

The results show that all cell populations produce antibodies. Additionof less than 10 nM MTX does not show much effect on the cells even atlow folic acid concentrations. The concentration of produced antibody iscomparable to selection in the absence of MTX. However, when using 10 nMMTX in the first selection step, productivity of cells at low folic acidconcentrations increase significantly and in a concentration dependentmanner. After selection with the lowest folic acid concentration (12.5nM) and 10 nM MTX, the productivity of the cells is found to be morethan 10 fold higher compared to medium with standard folic acidconcentration.

To further increase selection stringency, the next step is to removeG418 but to increase MTX concentration in the culture medium by a factorof 10, while keeping the folic acid concentration used in the firstselection step. In case of the reference, MTX is added at a highconcentration as is common in the prior art, here at 500 nM. Under theseconditions, viability of the cells of many transfected populationsdramatically drops and stays at low levels so that not all of them canbe recovered. Cell populations that could be recovered are furtherexpanded and productivity is analyzed (Tab. 2).

TABLE 2 Productivity of cell populations after 2nd selection step.Productivity Selection Medium mAb (mg/L Test series with 10 nM MTX andlow folic acid (FA) concentrations (12.5 nM, 25 nM and 50 nM) +w/o G418No recovery +12.5 nM FA +10 nM MTX +w/o G418 19 +25 nM FA +10 nM MTX+w/o G418 15 +50 nM FA +10 nM MTX Test series with 25 nM MTX and lowfolic acid (FA) concentrations (12.5 nM, 25 nM and 50 nM) +w/o G418 Norecovery +12.5 nM FA +25 nM MTX +w/o G418 No recovery +25 nM FA +25 nMMTX +w/o G418 20 +50 nM FA +25 nM MTX Test series with 50 mM MTX and lowfolic acid (FA) concentrations (12.5 nM, 25 nM and 50 nM) +w/o G418 Norecovery +12.5 nM FA +50 nM MTX +w/o G418 No recovery +25 nM FA +50 nMMTX +w/o G418 20 +50 nM FA +50 nM MTX Test series with 100 nM MTX andlow folic acid (FA) concentrations (12.5 nM, 25 nM and 50 nM) +w/o G418277 +12.5 nM FA +100 nM MTX +w/o G418 131 +25 nM FA +100 nM MTX +w/oG418 15 +50 nM FA +100 nM MTX Test series with non-limitingconcentration of folic acid (FA) (11.3 μM) and a MTX concentration of500 nM +w/o G418 31 +11.3 μM FA +500 nM MTX +w/o G418 26 +11.3 μM FA+500 nM MTX +w/o G418 21 +11.3 μM FA +500 nM MTX +w/o G418 25 +11.3 μMFA +500 nM MTX

G418 and MTX selected cell populations are further selected byincreasing the MTX concentration. Recovered populations are analyzed inshake flask batch cultures. At day 13 of the culture, samples of theculture medium are taken and analyzed for antibody content by Protein-AHPLC.

The productivity of the reference cell populations (500 nM MTX, 11.3 μMFA) after this selection step increase to approximately 25-30 mg/L. Nobenefit is seen at MTX concentrations below 100 nM. However, when using100 nM MTX in combination with a low folic acid content (12.5 or 25 nM)productivities are up to 277 mg/L and thus 10 times higher then thereference even though low MTX concentrations are used foramplification/selection.

Thus, using DHFR as selectable marker in combination with limiting folicacid concentration in the selective medium generates cells highlyoverexpressing a protein of interest even at low DHFR inhibitorconcentrations. The results also show that this combination is superiorto conventional selection systems (e.g. DHFR/G418 using standard folicacid concentration in the selective medium).

Example II Large Scale Production of Polypeptides with Transfected CHOCells

The production of polypeptides in large scale can be done for example inwave, glass or stainless steel bioreactors. For that purpose the cellsare expanded, usually starting from a single frozen vial, for example avial from a Master Cell Bank. The cells are thawed and expanded throughseveral steps. Bioreactors of different scale are inoculated withappropriate amounts of cells. The cell density can be increased byadding feed solutions and additives to the bioreactor. Cells are kept ata high viability for a prolonged time. Product concentrations in thereactor ranging from a few hundred milligrams per liter up to severalgrams per litre are achieved in the large scale. Purification can bedone by standard chromatography methodology, which can include affinity,ione exchange, hydrophobic interaction or size exclusion chromatographysteps. The size of the bioreactor can be up to several thousand litresvolume in the final scale (see also e.g. F. Wurm, Nature BiotechnologyVol. 22, 11, 2004, 1393-1398).

1. A method for selecting at least one eukaryotic host cell expressing aproduct of interest, comprising at least the following steps: (a)providing a plurality of eukaryotic host cells, wherein cellularviability of said host cells is dependent upon folate uptake, whereinsaid eukaryotic host cells comprise at least (i) an introducedpolynucleotide encoding a product of interest and (ii) an introducedpolynucleotide encoding a DHFR enzyme; (b) culturing said plurality ofeukaryotic host cells in a selective culture medium comprising at leastan inhibitor of DHFR and folate in a limiting concentration; (c)selecting at least one eukaryotic host cell expressing the product ofinterest.
 2. The method according to claim 1, wherein the selectiveculture medium comprises the DHFR inhibitor in a concentration of 500 nMor less and a folate in a concentration of 500 nM or less.
 3. The methodaccording to claim 1, wherein the selective culture medium comprises theDHFR inhibitor in a concentration of 200 nM or less and it comprises afolate in a concentration of 2.5 nM-100 nM.
 4. The method according toclaim 1 wherein the folate is folic acid in a concentration of 12.5nM-50 nM and wherein the DHFR inhibitor is MTX in a concentration of 10nM-100 nM.
 5. The method according to claim 1 wherein the DHFR enzyme isa DHFR enzyme having a lower sensitivity to a DHFR inhibitor than theDHFR enzyme endogenously expressed by the host cell.
 6. The methodaccording to claim 1 wherein said eukaryotic host cell is a CHO hostcell.
 7. The method according to claim 1 wherein a DHFR⁺ (plus) hostcell is used in conjunction with an introduced DHFR enzyme that is lesssensitive to MTX than the DHFR enzyme endogenously expressed by the hostcell.
 8. The method according to claim 1 wherein the polynucleotideencoding a product of interest and the polynucleotide encoding a DHFRenzyme have been introduced by at least one expression vector.
 9. Themethod according to, claim 1 wherein the host cell comprises at leasttwo introduced polynucleotides encoding a product of interest.
 10. Themethod according to claim 1, wherein the introduced polynucleotide(s)encoding the product of interest and the introduced polynucleotideencoding the DHFR enzyme are comprised in different expression cassettesand wherein the expression cassette(s) for driving the expression of thepolynucleotide(s) encoding the product of interest comprises a strongerpromoter and enhancer than the expression cassette for driving theexpression of the polynucleotide encoding the DHFR enzyme.
 11. Themethod according to claim 2 wherein the DHFR enzyme is a DHFR enzymehaving a lower sensitivity to a DHFR inhibitor than the DHFR enzymeendogenously expressed by the host cell.
 12. The method according toclaim 1 wherein said eukaryotic host cell is a DHFR⁺ (plus) cell. 13.The method according to claim 2 wherein a DHFR⁺ (plus) host cell is usedin conjunction with an introduced DHFR enzyme that is less sensitive toMTX than the DHFR enzyme endogenously expressed by the host cell. 14.The method according to claim 1 wherein the host cell comprises at leasttwo introduced polynucleotides encoding a product of interest, whereinat least one polynucleotide encodes the heavy chain of an immunoglobulinmolecule and the other polynucleotide encodes the light chain of animmunoglobulin molecule.
 15. The method according to claim 1 wherein theintroduced polynucleotide(s) encoding the product of interest and theintroduced polynucleotide encoding the DHFR enzyme are comprised indifferent expression cassettes and wherein the expression cassette(s)for driving the expression of the polynucleotide(s) encoding the productof interest comprises a stronger promoter or enhancer than theexpression cassette for driving the expression of the polynucleotideencoding the DHFR enzyme.